Sialic acids, such as N-acetylneuraminic acid (NANA), are .alpha.-ketoacids which occur as non-reducing terminal residues in the carbohydrate chains of glycoproteins and glycolipids. Abnormal levels of NANA have been detected in the serum and urine of cancer patients and of persons who are afflicted with certain congenital metabolic diseases. The early detection and quantitation of NANA thus offers a means of diagnosing various disease states and of providing early signals for the initiation of therapy.
The detection and measurement of sialic acid-terminated oligosaccharides has long presented a problem in biochemical investigations and routine analyses. The lack of detectable moieties which distinguish these molecules from other oligosaccharides has hindered developments in this area of glycobiology. Techniques for detection and quantitation of these molecules at early stages of their proliferation have been hampered by numerous problems, including lack of specificity of a derivatization reagent for .alpha.-keto acids or aldehydes, low sensitivity of the particular analytical technique employed, a lack of reagents which can be employed with highly polar analytes, and the instability of the derivatization reagents.
A spectrophotometric method of sialic acid determination using thiobarbituric acid has been employed, but this method lacks both selectivity and sensitivity. See, L. Warren, J. Biol. Chem. 234:1971 (1959) and D. Aminoff, Virology 7:355 (1959). Assays of NANA have also been performed as disclosed in the following publications: R. Schauer, Methods Enzymol. 50C:64 (1978), using gas chromatography (GC); I. Mononen, et al., FEBS Lett. 59:190 (1975), using gas chromatography-mass spectrometry (GC-MS); A. K. Shukla, et al., J. Chromatogr. 244:81 (1982) and H. K. B. Silver, et al., J. Chromatogr. 224:381 (1981), using high-performance liquid chromatography (HPLC) with UV detection; and S. Hara, et al., J. Chromatogr. 377:111-119 (1986), using HPLC with fluorescence detection. Both GC and HPLC with UV detection lack the sensitivity to detect NANA at picomole levels or below. Sensitivity is enhanced using GC-MS, but the cumbersome procedure for sample preparation limits the usefulness of this technique in rapid, diagnostic assays. The more recent method using HPLC with fluorescence detection involves the formation of a fluorescent quinoxaline from 1,2-diamino-4,5-dimethoxybenzene and NANA. This technique does not however permit the determination of highly polar analytes or provide for optimal reaction product behavior in electrophoresis and high-sensitivity fluorimetry. An additional problem is that the shelf life of the reagent employed is very limited.